IL-21 antagonists

ABSTRACT

Monoclonal antibodies are identified that bind the IL-21 protein. These antibodies are used to identify regions of the IL-21 protein to where binding neutralizes IL-21 activity. Hybridomas and methods of producing anti-IL-21 monoclonal antibodies are described. The monoclonal antibodies are useful in treating IL-21-mediated diseases, which may include autoimmune and inflammatory diseases such as pancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory bowel disease (IBD), Crohn&#39;s Disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren&#39;s syndrome, glomerulonephritis, IgA nephropathy, graft versous host disease, transplant rejection, atopic dermatitis, anti-phospholipid syndrome, and asthma, and other autoimmune diseases.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/179,003, filed Jul. 24, 2008, issued as U.S. Pat. No. 7,923,539,which is a divisional U.S. patent application Ser. No. 11/563,928, filedNov. 28, 2006, abandoned, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 60/740,154, filed Nov. 28, 2005, which areherein incorporated by reference.

BACKGROUND OF THE INVENTION

The immune system is the body's primary defense against diseases causedby pathogens, namely bacteria, viruses, fungi etc, as well as againstdiseases caused by abnormal growth of the body's own cells and tissues(i.e. cancerous tumors). Normally, the immune system is able todistinguish between the body's normal cells or “self” and foreignpathogens or abnormal cells or “non-self”. The processes by which theimmune system refrains from reacting to one's own body is calledtolerance. Sometimes, the immune system loses the ability to recognize“self” as normal and the subsequent response directed against the tissueor cells, results in loss of tolerance, a state of autoimmunity. Thepathologies resulting from autoimmunity often have serious clinicalconsequences and are one of the major health problems in the world,especially in developed nations.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. The interleukins are afamily of cytokines that mediate immunological responses. Receptors thatbind cytokines are typically composed of one or more integral membraneproteins that bind the cytokine with high affinity and transduce thisbinding event to the cell through the cytoplasmic portions of thecertain receptor subunits. Cytokine receptors have been grouped intoseveral classes on the basis of similarities in their extracellularligand binding domains. For example, the receptor chains responsible forbinding and/or transducing the effect of interferons are members of theclass II cytokine receptor family, based upon a characteristic 200residue extracellular domain.

The present invention provides anti-IL-21 monoclonal antibodies andmethods of using those antibodies that inhibit the symptoms andbiological activities that manifest as autoimmune and inflammatorydisorders and are associated with IL-21/IL-21 receptor interaction.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a bin or family ofantibodies that is capable of competition with a monoclonal antibodyproduced by the ATCC deposit 272.21.1.3.4.2 (ATCC Accession No.PTA-10395) for binding a human IL-21 antigen. In certain embodiments,the bin specifically binds to the epitope to which the monoclonalantibody produced by deposit 272.21.1.3.4.2 (ATCC Accession No.PTA-10395) binds.

In another aspect, the present invention provides a bin that is capableof competition with a monoclonal antibody produced by the ATCC deposit268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-10394) for binding a humanIL-21 antigen. In certain embodiments, the bin specifically binds to theepitope to which the monoclonal antibody produced by deposit268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-10394) binds.

In another aspect, the present invention provides for a hybridoma celldesignated ATCC deposit 272.21.1.3.4.2 (ATCC Accession No. PTA-10395;deposited Oct. 26, 2005; ATCC, 10801 University Blvd., Manassas, VA20110-2209) for producing a monoclonal antibody.

In another aspect, the present invention provides for a hybridoma celldesignated ATCC deposit 268.5.1.11.42.1.4.3.9 (ATCC Accession No.PTA-10394; deposited Oct. 26, 2005, ATCC, 10801 University Blvd.,Manassas, VA 20110-2209) for producing a monoclonal antibody.

In a further aspect, the present invention provides a method ofproducing the antibodies described herein comprising: (a) providing ahybridoma capable of producing the monoclonal antibody; and (b)culturing the hybridoma under conditions that provide for the productionthe monoclonal antibody by the hybridoma.

In one aspect, the present invention provides an anti-IL-21 monoclonalantibody that binds to an antigen region of human IL-21. In certainembodiments, the monoclonal antibody binds to an antigenic region ofIL-21 that is shown in SEQ ID NO: 6 from amino acid residues 97-122. Inanother embodiment, the monoclonal antibody binds an antigenic region asshown in SEQ ID NO: 6 from amino acid residues 145 to 148. In anotherembodiment, the monoclonal antibody binds an antigenic region as shownin SEQ ID NO: 6 from amino acid residues 154 to 162. In anotherembodiment, the monoclonal antibody binds an antigen region as shown inSEQ ID NO: 6 from amino acid residues 30 to 50. In another embodiment,the monoclonal antibody binds to an antigen region as shown in SEQ IDNO: 6 from amino acid residues 40 to 50. Additional embodiments includemonoclonal antibodies as described herein that can be shown toneutralize a human IL-21 protein activity, binds a human IL-21-Fcprotein, bind a human mutein Fc protein, where the mutations are at Gln145 and/or Ile148 of SEQ ID NO: 6, or bind a mouse IL-21-mouse Fc fusionprotein. Generally, the monoclonal antibodies of the present inventionbind two or more IL-21 proteins.

In other aspects, the monoclonal antibody specifically binds to theepitope to which monoclonal antibody produced by 272.21.1.3.4.2 (ATCCAccession No. PTA-10395) binds. In other embodiments, the monoclonalantibody specifically binds to the epitope to which monoclonal antibodyproduced by 268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-10394) binds.The monoclonal antibodies of the present invention may also be labeledwith a detectable marker and the detectable marker can be selected from,but is not limited to, radioactive isotopes, enzymes, dyes and biotins.

In another aspect, the present invention provides a bin (or group ofantibodies) that is capable of competition with monoclonal antibodyproduced by 272.21.1.3.4.2 (ATCC Accession No. PTA-10395) for binding ahuman IL-21 antigen.

Another aspect of the present invention provides a bin that is capableof competition with monoclonal antibody produced by268.5.1.11.42.1.4.3.9 (ATCC Accession No. PTA-10394) for binding a humanIL-21 antigen.

Also included in the present invention are hybridomas producing theclaimed monoclonal antibodies.

The present invention provides a method of producing the claimedmonoclonal antibodies comprising: (a) providing a hybridoma capapble ofproducing the monoclonal antibody; and (b) culturing the hybridoma underconditions that provide for production of the monoclonal antibody by thehybridoma.

In another aspect, the present invention provides a method of treatingan autoimmune disease comprising administering a therapeuticallyeffective amount of the claimed anti-IL-21 monoclonal antibodies to apatient. In certain embodiments, the autoimmune disease is selected fromthe group consisting of pancreatitis, type I diabetes (IDDM), GravesDisease, inflammatory bowel disease (IBD), Crohn's Disease, ulcerativecolitis, irritable bowel syndrome, multiple sclerosis, rheumatoidarthritis, diverticulosis, systemic lupus erythematosus, psoriasis,ankylosing spondylitis, scleroderma, systemic sclerosis, psoriaticarthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. hostdisease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome,glomerulonephritis, IgA nephropathy, graft versous host disease,transplant rejection, atopic dermatitis, anti-phospholipid syndrome, andasthma, and other autoimmune diseases.

The present invention also provides a method of inhibiting or reducingan IL-21-mediated disorder comprising administering an anti-IL-21monoclonal antibody in an amount sufficient to inhibit or reduce IL-21mediated biological activity in the subject.

DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate understanding ofthe inventions described herein.

The term “antibody” or “antibody peptide(s)” refers to an intactantibody, or a binding fragment thereof that competes with the intactantibody for specific binding and includes chimeric, humanized, fullyhuman, and bispecific antibodies. In certain embodiments, bindingfragments are produced by recombinant DNA techniques. In additionalembodiments, binding fragments are produced by enzymatic or chemicalcleavage of intact antibodies. Binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

The term “isolated antibody” refers to an antibody that has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, theantibody will be purified (1) to greater than 95% by weight of antibodyas determined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

A “variant” anti-IL-21 antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti-IL-21 antibody aminoacid sequence by virtue of addition, deletion and/or substitution of oneor more amino acid residue(s) in the parent antibody sequence. In thepreferred embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more hypervariable region(s) of the parentantibody. For example, the variant may comprise at least one, e.g. fromabout one to about ten, and preferably from about two to about five,substitutions in one or more hypervariable regions of the parentantibody. Ordinarily, the variant will have an amino acid sequencehaving at least 75% amino acid sequence identity with the parentantibody heavy or light chain variable domain sequences, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, and most preferably at least 95%. Identity or homology with respectto this sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical to the parentantibody residues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence shall be construed as affecting sequenceidentity or homology. The variant retains the ability to bind humanIL-21 and preferably has properties which are superior to those of theparent antibody. For example, the variant may have a stronger bindingaffinity, enhanced ability to inhibit IL-21-induced stimulation ofimmune cells. To analyze such properties, one should compare a Fab formof the variant to a Fab form of the parent antibody or a full lengthform of the variant to a full length form of the parent antibody, forexample, since it has been found that the format of the anti-IL-21antibody impacts its activity in the biological activity assaysdisclosed herein. The variant antibody of particular interest herein isone which displays at least about 10 fold, preferably at least about 20fold, and most preferably at least about 50 fold, enhancement inbiological activity when compared to the parent antibody.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule. IL-21 agonists cause, forexample: stimulation of NK cells, T cell subsets and B cell subsets anddendritic cells.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule. IL-21 antagonists cause:decreased immune function of NK cells, T cell subsets and B cell subsetsand dendritic cells; bind IL-21 such that the interaction of IL-21protein is blocked, inhibited, reduced, antagonized or neutralized.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol 79:315-321 (1990);Kostelny et al., J. Immunol. 148:1547-1553 (1992).

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used.

The term “effective neutralizing titer” as used herein refers to theamount of antibody which corresponds to the amount present in the serumof animals (human or cotton rat) that has been shown to be eitherclinically efficacious (in humans) or to reduce virus by 99% in, forexample, cotton rats. The 99% reduction is defined by a specificchallenge of, e.g., 10³ pfu, 10⁴ pfu, 10⁵ pfu, 10⁶ pfu, 10⁷ pfu, 10⁸pfu, or 10⁹ pfu) of RSV.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “IL-21 epitope” as usedherein refers to a portion of a IL-21 polypeptide having antigenic orimmunogenic activity in an animal, preferably in a mammal, and mostpreferably in a mouse or a human. An epitope having immunogenic activityis a portion of a IL-21 polypeptide that elicits an antibody response inan animal. An epitope having antigenic activity is a portion of a IL-21polypeptide to which an antibody immunospecifically binds as determinedby any method well known in the art, for example, by immunoassays.Antigenic epitopes need not necessarily be immunogenic.

The term “epitope tagged” when used herein refers to the anti-IL-21antibody fused to an “epitope tag”. The epitope tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere with activityof the IL-21 antibody. The epitope tag preferably is sufficiently uniqueso that the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu HA tagpolypeptide and its antibody 12CA5 (Field et al. Mol. Cell. Biol.8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (Paborsky et al., Protein Engineering 3(6):547-553 (1990)). Incertain embodiments, the epitope tag is a “salvage receptor bindingepitope”. As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃, or IgG₄) that is responsible for increasing the in vivoserum half-life of the IgG molecule.

The term “fragment” as used herein refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues of the amino acid sequence of a IL-21polypeptide or an antibody that immunospecifically binds to a IL-21polypeptide.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH₂-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk, J. Mol. Biol. 196: 901-917, 1987) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes and antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and (Fab)₂,as well as diabodies, linear antibodies, multivalent or multispecifichybrid antibodies (as described above and in detail in: Lanzavecchia etal., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Hustonet al., Proc. Natl. Acad. Sci. U.S.A., 85 5879-5883 (1988) and Bird etal., Science, 242:423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323:15-16 (1986),which are incorporated herein by reference).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding Single chain antibodies are discussed indetail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994); see also International Patent Application Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures ofwhich are incorporated by reference for any purpose. In specificembodiments, single-chain antibodies can also be bi-specific and/orhumanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof. Preferably, animmunologically functional immunoglobulin fragment of the inventionbinds specifically to IL-21.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

The present invention provides monoclonal antibodies and antibodyfragments that specifically bind with IL-21 proteins and polypeptides.Human and mouse IL-21 polypeptides, proteins and polynucleotidesencoding the polypeptides are disclosed in Parrish-Novak et al., Nature408:57-63, 2003, U.S. Pat. Nos. 6,307,024 and 6,686,178 and WO04/055168. Exemplary antibodies include neutralizing antibodies, and maybe murine monoclonal antibodies, humanized antibodies derived frommurine monoclonal antibodies, and human monoclonal antibodies.Illustrative antibody fragments include F(ab′)2, F(ab)₂, Fab′, Fab, Fv,scFv, and minimal recognition units. Neutralizing antibodies preferablybind IL-21 such that the interaction of IL-21 protein is blocked,inhibited, reduced, antagonized or neutralized. Described herein areepitopes and structural and functional characteristics defining regionsof the human IL-21 protein that have been identified as targets for atherapeutic monoclonal antibody. Exemplary mouse anti-human IL-21monoclonal antibodies and rat anti-human monoclonal antibodies and poolsof these monoclonal antibodies with the ability to bind wildtype humanIL-21, a mutant IL-21 protein and/or peptide regions of human IL-21 arepresented. The present invention further includes compositionscomprising a carrier and a peptide, polypeptide, or antibody describedherein.

Thus, the present invention provides that antagonists to IL-21 activity,such as anti-IL-21 antibodies, which are useful in therapeutic treatmentof inflammatory diseases. For example, anti-IL-21 antibodies are usefulin the treatment of pancreatitis, type I diabetes (IDDM), GravesDisease, inflammatory bowel disease (IBD), Crohn's Disease, ulcerativecolitis, irritable bowel syndrome, multiple sclerosis, rheumatoidarthritis, diverticulosis, systemic lupus erythematosus, psoriasis,ankylosing spondylitis, scleroderma, systemic sclerosis, psoriaticarthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. hostdisease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome,glomerulonephritis, IgA nephropathy, graft versous host disease,transplant rejection, atopic dermatitis, anti-phospholipid syndrome, andasthma, and other autoimmune diseases, or other diseases mediated byIL-21 and IL-21 receptor agonists.

The present invention also includes genetically altered antibodies thatare functionally equivalent to the above-described antibodies. Modifiedantibodies providing improved stability and/or therapeutic efficacy arepreferred. Examples of modified antibodies include those withconservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids which do not significantlydeleteriously alter the antigen binding utility. Substitutions can rangefrom changing or modifying one or more amino acid residues to completeredesign of a region as long as the therapeutic utility is maintained.Antibodies of the present invention can be can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).

The genetically altered antibodies also include chimeric antibodies thatderived from the anti-IL-21 antibodies. Preferably, the chimericantibodies comprise a variable region derived from a mouse or rat and aconstant region derived from a human so that the chimeric antibody has alonger half-life and is less immunogenic when administered to a humansubject. The method of making chimeric antibodies is known in the art.The variable regions of these antibodies can be connected with aconstant region of a human IgG to form the desired chimeric antibody.

Preferably, the genetically altered anti-IL-21 antibodies used in thepresent invention include humanized version of the antibodies describedherein. In certain embodiments, the humanized antibody comprising CDRsof a mouse donor immunoglobulin and heavy chain and light chainframeworks of a human acceptor immunoglobulin. The method of makinghumanized antibody is disclosed in U.S. Pat. Nos. 5,301,101; 5,585,089;5,693,762; and 6,180,370 (each of which is incorporated by reference inits entirety). The CDRs of these antibodies can then be grafted to anyselected human frameworks, which are known in the art, to generate thedesired humanized antibody.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, or by size in contiguous amino acidresidues. Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included.

Epitope binning refers to the use of competitive binding assays toidentify pairs of antibodies that are, or are not, capable of bindingIL-21 protein simultaneously thereby identifying antibodies that bind tothe same, or overlapping epitopes on protein. Families of antibodies (orbins) having the same binding specificity can then be used to definespecific epitopes on IL-21. Epitope binning experiments provide evidencethat antigenically distinct epitopes are present. However, bythemselves, they do not identify, or “map” the epitope to a specificamino acid sequence or location on the IL-21 protein molecule.

Competition for binding can be evaluated for any pair of antibodies orfragments. For example, using the appropriate detection reagents, thebinding specificity of antibodies or binding fragments from anyspecies/source can be compared to the binding specificity of themonoclonal antibodies disclosed herein. Epitope binning can be performedwith “isolated antibodies” or with cell culture supernatants. Frequentlybinning is performed with first round clonal supernatants to guide thechoice of clones to be developed further. The antibodies to be comparedshould have substantially homogeneous antigen binding domains. In thecase of “bispecific” or “bifunctional” antibodies the bindingspecificity of the two different binding sites need to be evaluated orbinned independently.

The present invention features both receptor-specific antibodies andligand-specific antibodies. In addition to competitive binding ofantibodies, epitope binning can also be used to identify antibodies toeither a receptor or a ligand that competitively interfere with thebinning of a ligand and its receptor. Frequently, favorable properties,of a family (or bin) of antibodies can correlated with a binding to aspecific epitope defined by the epitope bin.

Competitive binding experiments do not directly measure the bindingaffinity, however the antibodies to be tested must bind sufficientlystrongly to act as competitors. Generally experimental conditions aredesigned to minimize the effects of differences in binding affinity.

Anti-Antigen IL-21 antibodies may also be useful in diagnostic assaysfor IL-21 protein, e.g., detecting its expression in specific cells,tissues, or serum. Antibodies assigned to different bins and capable ofbinding to different immunogenic portions, or epitopes, of IL-21 may beused as the reagents for sandwich assays. In a sandwich assay, the testsample analyte is captured by a first antibody which is immobilized on asolid support, and thereafter detected by a second antibody that alsobinds to the analyte, thus forming an insoluble three-part complex. See,e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeledwith a detectable moiety (direct sandwich assays) or may be measuredusing an anti-immunoglobulin antibody that is labeled with a detectablemoiety (indirect sandwich assay). For example, one type of sandwichassay is an ELISA assay, in which case the detectable moiety is anenzyme.

The antibodies of the present invention may be assayed for specificbinding by any method known in the art. Many different competitivebinding assay format(s) can be used for epitope binning. Theimmunoassays which can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York). Exemplary immunoassays aredescribed briefly below (but are not intended by way of limitation).Additionally, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed.

The Biacore is only one of a variety of assay formats that are routinelyused to epitope bin panels of monoclonal antibodies. Many references(e.g. The Epitope Mapping Protocols, Methods in Molecular Biology,Volume 6.6 Glenn E. Morris ed.) describe alternative methods that couldbe used to bin antibodies and would be expected to provide identicalinformation regarding the binding specificity of the antibodies to IL-21protein. When using the Biacore system, epitope binning experiments areperformed with soluble, native antigen. Epitope binning studies can beperformed on a Biacore10000 system (Biacore, Uppsalla Sweden). BIAlogue®v. 1.2 software can be used for programming run methods. For the exampleof using the Biacore to bin mouse monoclonal antibodies raised againstIL-21, polyclonal goat anti-Mouse IgG Fc antibody (JacksonImmunoResearch Laboratories, West Grove, Pa.) can be covalentlyimmobilized to a Biacore® CM5 sensor chip and used to bind (capture) theprimary monoclonal antibody of test series to the chip. Unoccupied Fcbinding sites on the chip are then blocked using a polyclonal IgG Fcfragment (Jackson ImmunoResearch Laboratories, West Grove, Pa.).Subsequently, IL-21 protein is injected and allowed to specifically bindto the captured primary monoclonal antibody. The Biacore instrumentmeasures the mass of protein bound to the sensor chip, and the bindingof both the primary antibody and IL-21 antigen can be verified for eachcycle. Following the binding of the primary antibody and antigen to thechip, soluble secondary antibody is injected and allowed to bind to thepre-bound antigen. If the secondary monoclonal antibody is capable ofbinding the IL-21 antigen simultaneously with the primary monoclonalantibody, its binding is detected by the Biacore. If, however, thesecondary monoclonal antibody is not capable of binding the IL-21antigen simultaneously with the primary monoclonal antibody, noadditional binding is detected. Each monoclonal antibody is testedagainst itself as a negative control to establish the level of thebackground (no-binding) signal.

A label-free competitive ELISA format (LFC-ELISA) can also be used tobin antibodies. This method is described by Nagata et al., J. ImmunoMethods 292:141-155, 2004. This method for epitope binning utilizedbiotinylated IL-21. For the example of binning mouse monoclonalantibodies raised against IL-21, microtiter plates are coated at 100μL/well with 1 μg/mL of a goat anti-mouse IgG Fc-γ specific antibody(Jackson ImmunoResearch) diluted in ELISA B (PBS, 0.1% Tween 20, 1%BSA). After binding of this coating antibody for 3 hours at ambienttemperature, each mAb-containing conditioned media is diluted in ELISA Bto yield an approximate mAb concentration of 0.5 μg/mL and allowed tobind to the goat anti-mouse IgG coated plates overnight at 4° C.(mAb#1). In parallel, a second set of conditioned medias (mAb#2) arediluted in polystyrene test tubes to approximately 0.5 μg/mL mAb inELISA B, mixed with 50 ng/mL biotinylated IL-21 antigen, and incubatedovernight at 4° C. After incubation of mAb#1 with the coating antibody,the plates are blocked with an unrelated antibody to saturate unoccupiedbinding sites on the plate. The mAb#2-biotin-IL-21 mixtures are added tothe plate and allowed to bind. As a control for (non-competition) in theassay, 50 ng/mL biotinylated IL-21 is added directly (withoutpre-incubation with mAb#2) to wells containing immobilized mAb#1. Afterincubation with the biotinylated-IL-21-mAb#2 complex, streptavidin-HRP(Pierce, Rockford, Ill.) is added to the plate at 0.5 μg/mL. The platesare developed with TMB substrate (BioFX Laboratories, Owings Mills,Md.), and the absorbance of the individual wells at 450 nm is measuredwith a plate reader (Molecular Devices SpectraMax®340, Sunnyvale,Calif.). If mAb#1 binds to a different epitope from mAb#2, thebiotin-IL-21-mAb#2 complex will bind to the plate resulting in a highabsorbance reading. If mAb#1 binds to the same epitope as mAb#2, thebiotin-IL-21-MAb#2 complex will not bind to the plate resulting in a lowabsorbance reading.

Antibodies of the present invention act as antagonists of IL-21. Forexample, the present invention includes antibodies which disrupt IL-21'sreceptor/ligand interactions either partially or fully. The inventionfeatures ligand-specific antibodies that prevent receptor activation.The invention includes neutralizing antibodies which bind the ligand andprevent binding of the ligand to the receptor, as well as antibodieswhich bind the ligand, thereby preventing receptor activation, but donot prevent the ligand from binding the receptor. Receptor activation(i.e., signaling) may be determined by techniques described herein orotherwise known in the art. For example, receptor activation can bedetermined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot or luminex based analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

Production of Anti-IL-21 Antibodies

Antibodies to IL-21 can be obtained, for example, using the product of aIL-21 expression vector or IL-21 isolated from a natural source as anantigen. Particularly useful anti-IL-21 antibodies “bind specifically”with IL-21. Antibodies are considered to be specifically binding if theantibodies exhibit at least one of the following two properties: (1)antibodies bind to IL-21 with a threshold level of binding activity, and(2) antibodies do not significantly cross-react with polypeptidesrelated to IL-21.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a IL-21 polypeptide, peptide or epitope with a bindingaffinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, morepreferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater.The binding affinity of an antibody can be readily determined by one ofordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 1949) or using a commerciallyavailable biosensor instrument (BIAcore, Pharmacia Biosensor,Piscataway, N.J.). With regard to the second characteristic, antibodiesdo not significantly cross-react with related polypeptide molecules, forexample, if they detect IL-21, but not other known polypeptides using astandard Western blot analysis or capture ELISA. Examples of knownrelated polypeptides include known members of the IL-2 family.

Anti-IL-21 antibodies can be produced using antigenic IL-21epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2 or another amino acid sequence disclosed herein. However,peptides or polypeptides comprising a larger portion of an amino acidsequence of the invention, containing from 30 to 50 amino acids, or anylength up to and including the entire amino acid sequence of apolypeptide of the invention, also are useful for inducing antibodiesthat bind with IL-21. It is desirable that the amino acid sequence ofthe epitope-bearing peptide is selected to provide substantialsolubility in aqueous solvents (i.e., the sequence includes relativelyhydrophilic residues, while hydrophobic residues are typically avoided).Moreover, amino acid sequences containing proline residues may be alsobe desirable for antibody production.

Monoclonal anti-IL-21 antibodies can be generated by methods known tothose skilled in the art. Rodent monoclonal antibodies to specificantigens may be obtained by known methods (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Selection of binders from the display of a library of antibody fragmentsis an in vitro alternative to the development of monoclonal antibodies.The principle of display technology is establishment of a physicalconnection between a binding moiety and the encoding genetic material.This concept has been used in a number of modes from display of proteinand peptide libraries on surfaces of bacteriophage, bacteria, and yeastto the display of proteins attached to ribosomes in vitro (see forexample Rothe et al., FASEB J. 20:1599 (2006)). The display ofantibodies on the surface on single-stranded bacteriophage is the mosthighly developed of these technologies. The typical method used forantibody display is to fuse either the single chain Fv fragment or theheavy chain Fd (heavy chain portion of a Fab) with the gene III proteinof the phage. Antibody libraries can be naïve, representing the naturalimmune repertoire, or semi-synthetic, consisting of frameworks takenfrom native human templates combined with synthetic CDR sequencelibraries to increase diversity. Phage with specific binding activitiescan be isolated from random libraries of antibody fragments(particularly Fab and scFv) or peptides after repeated rounds of growthand selection (see, for example, Hoogenboom, Nature Biotech. 23:1105(2005.)

In a further embodiment, antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety. In yet a further embodiment, antibodies orantibody fragments can be isolated from antibody phage librariesgenerated using the techniques described in McCafferty et al., Nature,348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) andMarks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolationof murine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity (nMrange) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10: 779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869,1992; and Sawai et al., AJRI 34:26-34, 1995; and Better et al., Science240:1041-1043, 1988 (all references incorporated by reference in theirentireties).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (Int. Rev. Immunol 13:65-93, 1995).

For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); PCT publications WO98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,which are incorporated by reference herein in their entirety. Inaddition, companies such as Medarex, Inc. (Princeton, N.J.) and Genpharm(San Jose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove. See, e.g. U.S. Pat. No. 7,135,287.

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., MPSV, CMV, the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus, CMV enhancer or MPSV promoter is an effectiveexpression system for antibodies (Foecking et al., 1986, Gene 45:101;Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109, 1985; Van Heeke & Schuster, J. Biol. Chem.24:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359, 1984). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., Methods in Enzymol.153:51-544, 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202, 1992), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes can beemployed in tk−, hgprt− or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:357, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418 (Wu andWu, Biotherapy 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596, 1993; Mulligan, Science 260:926-932, 1993; and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217, 1993; TIB TECH 11(5):155-215),May, 1993; and hygro, which confers resistance to hygromycin (Santerreet al., Gene 30:147, 1984). Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.(eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons,NY.; Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981; which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257,1983).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, Nature 322:52, 1986; Kohler, Proc. Natl. Acad. Sci. USA77:2197, 1980). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

For particular uses, it may be desirable to prepare fragments ofanti-IL-21 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)2. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960; Porter, Biochem.J. 73:119, 1959; Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of VH and VL chains.This association can be noncovalent, as described by Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chainscan be linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev.Biotech. 12:437, 1992).

The Fv fragments may comprise VH and VL chains which are connected by apeptide linker. These single-chain antigen binding proteins (scFv) areprepared by constructing a structural gene comprising DNA sequencesencoding the VH and VL domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271, 1993, and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toIL-21 polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled IL-21 protein or peptide). Genes encoding polypeptides havingpotential IL-21 polypeptide binding domains can be obtained by screeningrandom peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the IL-21 sequences disclosed herein to identifyproteins which bind to IL-21.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-IL-21 antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse or rat complementary determining regions from heavyand light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833, 1989. Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285,1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; Singer et al., J. Immun.150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762.

It is also possible to construct alternative frameworks by using acollection of monomeric proteins to form a monomer domain. These monomerdomains can be small enough to penetrate tissues. The monomer domainscan be naturally-occurring or non-natural vaiants or combinationthereof. Monomer domains can form multimers of two more domains. Themonomer domain binds a position, analogous to epitopes described herein,on a target molecule. In some cases, the multimer can be formed fromvariety of monomer domains. (See, e.g. U.S. Patent Application2004-0132028 and U.S. Patent Application 2006-0177831.)

The antibodies of the present invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom binding IL-21 or preventing receptor activation. For example, butnot by way of limitation, the antibody derivatives include antibodiesthat have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

An anti-IL-21 antibody can be conjugated with a detectable label to forman anti-IL-21 immunoconjugate. Suitable detectable labels include, forexample, a radioisotope, a fluorescent label, a chemiluminescent label,an enzyme label, a bioluminescent label or colloidal gold. Methods ofmaking and detecting such detectably-labeled immunoconjugates arewell-known to those of ordinary skill in the art, and are described inmore detail below. The detectable label can be a radioisotope that isdetected by autoradiography. Isotopes that are particularly useful forthe purpose of the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-IL-21 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

It is also possible that anti-IL-21 immunoconjugates can be detectablylabeled by coupling an antibody component to a chemiluminescentcompound. The presence of the chemiluminescent-tagged immunoconjugate isdetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of chemiluminescent labelingcompounds include luminol, isoluminol, an aromatic acridinium ester, animidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-IL-21immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-IL-21 immunoconjugates can be detectably labeled bylinking an anti-IL-21 antibody component to an enzyme. When theanti-IL-21-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-IL-21 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1, 1976; Schurs etal., Clin. Chim. Acta 81:1, 1977; Shih et al., Int'l J. Cancer 46:1101,1990; Stein et al., Cancer Res. 50:1330, 1990; and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-IL-21 antibodies that have been conjugatedwith avidin, streptavidin, and biotin (see, for example, Wilchek et al.(eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184(Academic Press 1990), and Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10,Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

Antibodies or fragments thereof having increased in vivo half-lives canbe generated by techniques known to those of skill in the art. Forexample, antibodies or fragments thereof with increased in vivohalf-lives can be generated by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631 and WO 02/060919, which are incorporatedherein by reference in their entireties). Antibodies or fragmentsthereof with increased in vivo half-lives can be generated by attachingto said antibodies or antibody fragments polymer molecules such as highmolecular weight polyethyleneglycol (PEG). PEG can be attached to saidantibodies or antibody fragments with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or antibody fragments or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation will be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by, e.g., size exclusion or ion-exchange chromatography.

Pharmaceutical Compositions

The present invention further includes pharmaceutical compositions,comprising a pharmaceutically acceptable carrier and a polypeptide orantibody described herein. The pharmaceutical composition can includeadditional therapeutic agents, including but not limited to cytotoxicagents a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeuticagent or a radioactive metal ion. A cytotoxin or cytotoxic agentincludes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). For example, the pharmaceuticalcomposition can comprise a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-IFN, β-IFN, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

For purposes of therapy, anti-IL-21 antibody molecules and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a therapeuticmolecule of the present invention and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates the inflammatory response.

A pharmaceutical composition comprising anti-IL-21 antibody can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions and oral suspensions. Exemplarysolid forms include capsules, tablets, and controlled-release forms. Thelatter form is illustrated by miniosmotic pumps and implants (Bremer etal., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants in DrugDelivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.), pages95-123 (CRC Press 1995); Bremer et al., “Protein Delivery with InfusionPumps,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 239-254 (Plenum Press 1997); Yewey et al., “Delivery ofProteins from a Controlled Release Injectable Implant,” in ProteinDelivery Physical Systems, Sanders and Hendren (eds.), pages 93-117(Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):561 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287, 1997; Murahashi et al., Biol. Pharm.Bull. 20:259, 1997). Similarly, Wu et al., Hepatology 27:772, 1998, haveshown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259, 1997). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681, 1997).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99, 1998).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., ibid. (1998)).

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099, 1981, Anderson et al., CancerRes. 50:1853, 1990, and Cohen et al., Biochim. Biophys. Acta 1063:95,1991, Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124,1987). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9, 1993).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161, 1998; Putney andBurke, Nature Biotechnology 16:153, 1998; Putney, Curr. Opin. Chem.Biol. 2:548, 1998). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167,1997).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5th Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises a neutralizing anti-IL-21 antibody).Therapeutic polypeptides can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition.

A pharmaceutical composition comprising anti-IL-21 antibodies can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions, aerosols, droplets, topologicalsolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.10:239, 1997; Ranade, “Implants in Drug Delivery,” in Drug DeliverySystems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995);Bremer et al., “Protein Delivery with Infusion Pumps,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254(Plenum Press 1997); Yewey et al., “Delivery of Proteins from aControlled Release Injectable Implant,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).Other solid forms include creams, pastes, other topologicalapplications, and the like.

Therapeutic Uses for Anti-IL-21 Antibodies

IL-21 is a CD4⁺ T cell-derived cytokine that is important for optimalCD8⁺ T cell mediated immunity, NK cell activation, and optimal humoralresponses, such as antibody production and B cell maturation. IL-21 hasbeen shown to induce a number of proinflammatory chemokines andcytokines, such as IL-18, IL-15, IL-5, IL-6, TNFR11, sCD25, and RANTES.IL-21 also induces an acute phase response in non-human primates andhumans. Increased expression of IL-21 receptor has been shown inepidermis in patients with systemic sclerosis (Distler et al., Arthritis& Rheumatism 52:865-864, 2004) and rheumatoid arthritis synovialfibroblasts (Jungel et al., Arthritis & Rheumatism 50:1468-1476, 2004).Moreover, autoimmune, diabetic NOD mice have increased IL-21 receptorexpression (King et al., Cell 117:265-277, 2004.) It has been shown thatIgG and IL-21 expression is increased in the BXSB-Yaa mouse model whichdevelop an autoimmune lupus erythematosus-like disease (Ozaki et al., J.Immunol. 173:5361-5371, 2004); IL-21 expression is higher in lupus-proneSanroque mice (Vinuesa et al. Nature 435:452, 2005); IL-21 expression ishigher in patients with Crohn's disease (Monteleone, et al.,Gastroenterology 128:687-694, 2005).

A therapeutically effective amount of an anti-IL-21 antibody refers toan amount of antibody which when administered to a subject is effectiveto prevent, delay, reduce or inhibit a symptom or biological activityassociated with a disease or disorder. Administration may consist of asingle dose or multiple doses and may be given in combination with otherpharmaceutical compositions.

The present invention provides compositions and methods for using IL-21antagonists in inflammatory and immune diseases or conditions such aspancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory boweldisease (IBD), Crohn's Disease, ulcerative colitis, irritable bowelsyndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis,systemic lupus erythematosus, psoriasis, ankylosing spondylitis,scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis,atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous Tcell lymphoma (CTCL), Sjogren's syndrome, glomerulonephritis, IgAnephropathy, graft versous host disease, transplant rejection, atopicdermatitis, anti-phospholipid syndrome, and asthma, and other autoimmunediseases.

Contact Dermatitis

Allergic contact dermatitis is defined as a T cell mediated immunereaction to an antigen that comes into contact with the skin. The CLA+ Tcell population is believed to be involved in the initiation ofdermatitis since allergen dependent T cell responses are largelyconfined to the CLA+ population of cells (See Santamaria-Babi, L. F., etal., J Exp Med 181:1935, (1995)). Recent data have found that onlymemory (CD45RO+) CD4+ CLA+ and not CD8+ T cells proliferate and produceboth type-1 (IFN-γ) and type-2 (IL-5) cytokines in response to nickel, acommon contact hypersensitivity allergen. Furthermore, cells expressingCLA in combination with CD4, CD45RO (memory) or CD69 are increased afternickel-specific stimulation and express the chemokine receptors CXCR3,CCR4, CCR10 but not CCR6. See Moed H., et al., Br J Dermatol 51:32,(2004).

In animal models, it has been demonstrated that allergic contactdermatitis is T cell-dependent and that the allergic-responsive T cellsmigrate to the site of allergen application. See generally: Engeman T.M., et al., J Immunol 164:5207, (2000); Ferguson T. A. & Kupper T. S. JImmunol 150:1172, (1993); and Gorbachev A. V. & Fairchild R. L. Crit RevImmunol 21:451 (2001).

Atopic Dermatitis

Atopic dermatitis (AD) is a chronically relapsing inflammatory skindisease with a dramatically increasing incidence over the last decades.Clinically AD is characterized by highly pruritic, often excoriated,plaques and papules that show a chronic relapsing course. The diagnosisof AD is mostly based on major and minor clinical findings. See HanifinJ. M., Arch Dermatol 135:1551 (1999). Histopathology reveals spongiosis,hyperparakeratosis and focal parakeratosis in acute lesions, whereasmarked epidermal hyperplasia with hyperparakeratosis and parakeratosis,acanthosis/hypergranulosis and perivascular infiltration of the dermiswith lymphocytes and abundant mast cells are the hallmarks of chromiclesions.

T cells play a central role in the initiation of local immune responsesin tissues and evidence suggests that skin-infiltrating T cells inparticular, may play a key role in the initiation and maintenance ofdisregulated immune responses in the skin. Approximately 90% ofinfiltrating T cells in cutaneous inflammatory sites express thecutaneous lymphocyte-associated Ag (CLA+) which binds E-selectin, aninducible adhesion molecule on endothelium (reviewed in Santamaria-BabiL. F., et al., Eur J Dermatol 14:13, (2004)). A significant increase incirculating CLA+ T cells among AD patients compared with controlindividuals has been documented (See Teraki Y., et al., Br J Dermatol143:373 (2000), while others have demonstrated that memory CLA+ T cellsfrom AD patients preferentially respond to allergen extract compared tothe CLA-population (See Santamaria-Babi, L. F., et al., J Exp Med.181:1935, (1995)). In humans, the pathogenesis of atopic disorders ofthe skin have been associated with increases in CLA+ T cells thatexpress increased levels of Th-2-type cytokines like IL-5 and IL-13. SeeAkdis M., et al., Eur J Immunol 30:3533 (2000); and Hamid Q., et al., JAllergy Clin Immunol 98: 225 (1996).

NC/Nga mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histophathologyand immunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka H., et al., Allergy 58:139 (2003).Therefore, the development of AD in NC/Nga is a useful model for theevaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel J.M., et al., J Clin Invest, 101:1614, (1998). This protocol can bemodified in order to induce skin barrier disregulation and pruritis bysensitizing DO11.10 OVA TCR transgenic mice with OVA. Increasing thenumber of antigen-specific T cells that could recognize the sensitizingantigen may increase the level of inflammation in the skin to inducevisible scratching behaviour and lichenification/scaling of the skin.

Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory antibodies and binding polypeptides. For example,rheumatoid arthritis (RA) is a systemic disease that affects the entirebody and is one of the most common forms of arthritis. It ischaracterized by the inflammation of the membrane lining the joint,which causes pain, stiffness, warmth, redness and swelling. Inflammatorycells release enzymes that may digest bone and cartilage. As a result ofrheumatoid arthritis, the inflamed joint lining, the synovium, caninvade and damage bone and cartilage leading to joint deterioration andsevere pain amongst other physiologic effects. The involved joint canlose its shape and alignment, resulting in pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

The administration of anti-IL-21 antibodies to these CIA model mice areused to evaluate the use of anti-IL-21 antibodies to ameliorate symptomsand alter the course of disease.

Inflammatory Bowel Disease (IBD)

In the United States approximately 500,000 people suffer frominflammatory bowel disease (IBD) which can affect either colon andrectum (ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Ulcerative colitis(UC) is an inflammatory disease of the large intestine, commonly calledthe colon, characterized by inflammation and ulceration of the mucosa orinnermost lining of the colon. This inflammation causes the colon toempty frequently, resulting in diarrhea. Symptoms include loosening ofthe stool and associated abdominal cramping, fever and weight loss.Although the exact cause of UC is unknown, recent research suggests thatthe body's natural defenses are operating against proteins in the bodywhich the body thinks are foreign (an “autoimmune reaction”). Perhapsbecause they resemble bacterial proteins in the gut, these proteins mayeither instigate or stimulate the inflammatory process that begins todestroy the lining of the colon. As the lining of the colon isdestroyed, ulcers form releasing mucus, pus and blood. The diseaseusually begins in the rectal area and may eventually extend through theentire large bowel. Repeated episodes of inflammation lead to thickeningof the wall of the intestine and rectum with scar tissue. Death of colontissue or sepsis may occur with severe disease. The symptoms ofulcerative colitis vary in severity and their onset may be gradual orsudden. Attacks may be provoked by many factors, including respiratoryinfections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of anti-IL-21 antibodies to these TNBS, DSS or CD4transfer models can be used to evaluate the use of IL-21 antagonists toameliorate symptoms and alter the course of gastrointestinal disease.IL-21 may play a role in the inflammatory response in colitis, and theneutralization of IL-21 activity by administrating IL-21 antagonists isa potential therapeutic approach for IBD.

Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. Anti-IL-21antibodies of the present invention, could serve as a valuabletherapeutic to reduce inflammation and pathological effects inpsoriasis, other inflammatory skin diseases, skin and mucosal allergies,and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy. Anti-IL-21 antibodies can be testedusing a recently developed a model of psoriasis based on the CD4+CD45RBtransfer model (Davenport et al., Internat. Immunopharmacol., 2:653-672,2002).

In addition to other disease models described herein, the activity ofanti-IL-21 antibodies on inflammatory tissue derived from humanpsoriatic lesions can be measured in vivo using a severe combined immunedeficient (SCID) mouse model. Several mouse models have been developedin which human cells are implanted into immunodeficient mice(collectively referred to as xenograft models); see, for example, CattanA R, Douglas E, Leuk. Res. 18:513-22, 1994 and Flavell, D J,Hematological Oncology 14:67-82, 1996. As an in vivo xenograft model forpsoriasis, human psoriatic skin tissue is implanted into the SCID mousemodel, and challenged with an appropriate antagonist. Moreover, otherpsoriasis animal models in ther art may be used to evaluate IL-21antagonists, such as human psoriatic skin grafts implanted into AGR129mouse model, and challenged with an appropriate antagonist (e.g., see,Boyman, O. et al., J. Exp. Med. Online publication #20031482, 2004,incorporated hereing by reference). Similarly, tissues or cells derivedfrom human colitis, IBD, arthritis, or other inflammatory lestions canbe used in the SCID model to assess the anti-inflammatory properties ofthe anti-IL-21 antibodies described herein.

Efficacy of treatment is measured and statistically evaluated asincreased anti-inflammatory effect within the treated population overtime using methods well known in the art. Some exemplary methodsinclude, but are not limited to measuring for example, in a psoriasismodel, epidermal thickness, the number of inflammatory cells in theupper dermis, and the grades of parakeratosis. Such methods are known inthe art and described herein. For example, see Zeigler, M. et al. LabInvest 81:1253, 2001; Zollner, T. M. et al. J. Clin. Invest. 109:671,2002; Yamanaka, N. et al. Microbio.l Immunol 45:507, 2001; Raychaudhuri,S. P. et al. Br. J. Dermatol. 144:931, 2001; Boehncke, W. H et al. Arch.Dermatol. Res. 291:104, 1999; Boehncke, W. H et al. J. Invest. Dermatol.116:596, 2001; Nickoloff, B. J. et al. Am. J. Pathol. 146:580, 1995;Boehncke, W. H et al. J. Cutan. Pathol. 24:1, 1997; Sugai, J., M. et al.J. Dermatol. Sci. 17:85, 1998; and Villadsen L. S. et al. J. Clin.Invest. 112:1571, 2003. Inflammation may also be monitored over timeusing well-known methods such as flow cytometry (or PCR) to quantitatethe number of inflammatory or lesional cells present in a sample, score(weight loss, diarrhea, rectal bleeding, colon length) for IBD, pawdisease score and inflammation score for CIA RA model.

Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an immune-complex related disordercharacterized by chronic IgG antibody production directed at ubiquitousself antigens (e.g. anti-dsDNA). The effects of SLE are systemic, ratherthan localized to a specific organ. Multiple chromosomal loci have beenassociated with the disease and may contribute towards different aspectsof the disease, such as anti-dsDNA antibodies and glomerulonephritis.CD4+ T cells have been shown to play an active part in mouse models ofSLE (Horwitz, Lupus 10:319-320, 2001; Yellin and Thienel, Curr.Rheumatol. Rep., 2:24-37, 2000). The role for CD8+ T cells is notclearly defined, but there is evidence to suggest that “suppressor” CD8+T cell function is impaired in lupus patients (Filaci et al., J.Immunol., 166:6452-6457, 2001; Sakane et al, J. Immunol., 137:3809-3813,1986).

IL-21 has been shown to modulate antibody responses by directly actingon B cells. (Mehta et al., J. Immunol, 170:4111-4118, 2003; Ozaki etal., Science, 298:1630-1634, 2002; Suto et al., Blood, 100:4565-4573,2002). For example, Ozaki et al., (J. Immunol. 173:5361, 2004)demonstrated that in BXSB-Yaa mice, a model for SLE, there is anelevated serum IL-21 level. Moreover, because IL-21 enhances CD8⁺ T cellactivity, administration of anti-IL-21 antibodies would provide a morerobust T cell suppressor function in lupus patients where that functionis compromised.

Anti-IL-21 antibodies can be administered in combination with otheragents already in use in autoimmunity including immune modulators suchas IFNγ, NOVANTRONE®, ENBREL®, REMICADE®, LEUKINE® and IL-2.Establishing the optimal dose level and scheduling for anti-IL-21antibodies is done by a variety of means, including study of thepharmacokinetics and pharmacodynamics of anti-IL-21 antibodies;determination of effective doses in animal models, and evaluation of thetoxicity of anti-IL-21 antibodies. Direct pharmacokinetic measurementsdone in primates and clinical trials can then be used to predicttheoretical doses in patients that achieve plasma anti-IL-21 antibodylevels that are of sufficient magnitude and duration to achieve abiological response in patients.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Preparation of IL-21 Proteins

IL-21 protein was produced as described in U.S. Patent Application No.2006-0134754 and WO 04/055168, incorporated in its entirety herein.Briefly, a IL-21 nucleotide sequence was optimized and inserted in an E.coli expression vector which was deposited as ATCC Accession No.PTA-4853. The expression vector was introduced into E. coli strain W3110(ATCC Accession No. 27325).

Host cells were fermented by growing E. coli strains expressing IL-21 ina suitable medium in shake flask culture to in a suitable medium and maybe supplemented with carbohydrates, such as fructose, glucose,galactose, lactose, and glycerol. Isopropyl thiogalactopyranoside (IPTG)is may be added to the culture to a concentration 0.1 to 2.0 mM.

Following fermentation the cells were harvested by centrifugation,re-suspended in homogenization buffer and homogenized. After thehomogenate was collected, it was resuspended a guanidine containingsolution and the supernatant containing solubilized IL-21 was decantedand retained. The concentration of the IL-21 in the solubilized fractionwas determined by reversed phase HPLC. Once the inclusion bodies weresolubilized and denatured in guanidine solution containing a reducingagent, the reduced IL-21 was then oxidized in a controlled renaturationstep. This step involved dilution in a refold buffer containing argininehydrochloride, salts, and an oxido-shuffling system.

Purification of IL-21 protein may include purification of the IL-21using hydrophobic interaction chromatography. The IL-21 may be furtherpurified by high performance cation exchange chromatography. The methodsfor purifying IL-21 can comprise concentrating and carrying out a bufferexchange of the protein. This step is designed to concentrate the highperformance cation exchange column eluate and exchange it intoformulation buffer. The final column eluate pool is concentrated toincrease the concentration of IL-21. Further purification of IL-21 toremove the remaining impurities and contaminants may be desirable. Forexample, an anion exchange column can be used to reduce the endotoxinlevel.

Example 2 Preparation of IL-21 Receptor Proteins

The IL-21 receptor (also designated as zalpha11 or IL-21r) heterodimerprotein can be produced as described in U.S. Patent Application No.2002-0137677, incorporated in its entirety herein. Briefly, a vectorexpressing a secreted human hzalpha11/hIL2Rgamma heterodimer isconstructed. In this construct, the extracellular domain of hzalpha11 isfused to the CH1 domain of IgG γ1. The CH1 domain is cloned into amammalian expression vector. The CL1 domain of the human K light chainis cloned in a mammalian expression vector.

A construct having human zalpha11 fused to CH₁ is made, and the vectoris sequenced to confirm that the fusion is correct. A separate constructhaving hIL2Rgamma fused to CL1 can be also constructed. The resultingvector is sequenced to confirm that the human IL-2Rgamma/CL1 fusion iscorrect.

The human zalpha11 (IL-21r) and human IL-2Rgamma receptor fusions areco-expressed. Each expression vector is co-transfected into mammalianhost cells by methods known to those skilled in the art. The transfectedcells are selected for 10 days in methotrexate (MTX) and G418(Gibco/BRL) for 10 days. The resulting pool of transfectants is selectedagain in MTX and G418 for 10 days.

The resulting pool of doubly-selected cells is used to generate protein.Factories (Nunc, Denmark) of this pool are used to generate conditionedmedium. This serum free, conditioned media is passed over a protein-Acolumn and eluted in fractions. Fractions found to have the highestconcentration are pooled and dialyzed (10 kD MW cutoff) against PBS.Finally the dialyzed material is submitted for amino acid analysis(AAA). The purified soluble human zalpha11 receptor/IL-2Rgamma receptorcan be used to assess its ability to compete for binding of the humanzalpha11 Ligand a BaF3 proliferation assay.

B. The extracellular domain of human zalpha11 fused to Fc9 (Fc region ofhuman gamma1 (Kabat numbering 221-447; Kabat et al, Sequences ofProteins of Immunological Interest, U.S. Dept. Health and Human Serv.,Bethesa, Md., 1991)) with an GluGlu tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:7952-4, 198)) at the carboxyl terminus wasgenerated by overlap PCR. The cDNA was inserted into pZMP31 (describedin US Patent application, US2003/023414; a hybrid vector having acytomegalovirus enhancer and myeloproliferative sarcoma virus promoter)by recombination in yeast. The extracellular domain of the human IL2receptor common gamma chain was fused to Fc9 with a 6×His tag at thecarboxyl terminus of Fc9. This construct was inserted into pZMP21z byyeast recombination using the same method as described for zalpha11Fc9CEE. The resulting constructs were sequenced to verify that theinserts were correct. Both plasmids were transfected into suspension,serum-free-adapted CHO cells by electroporation and selected inprotein-free PFCHO media (BioWhittaker) without hypoxanthine andthymidine with 200 ng/mL zeomycin added. These cells were then selectedin the same medium plus increasing concentrations of methotrexate untilthe cells were resistant to both 1 uM methotrexate and 200 ng/mLzeomycin. The cells were tested for production of heterodimeric IL21receptor by western blot analysis for the presence of both EE and histags.

The design of zcytor26f2 (extracellular domain of the human IL2 receptorcommon gamma chain was fused to Fc9 with a 6×His tag) is such that threetags are available for purification (GluGlu, His, and Fc), of which twoare utilized to best discriminate heterodimer from the two homodimercontaminants. All molecules containing an Fc domain (homodimercontaminants and heterodimer target) were captured and purified fromhost cell components and related media products. The pool containing allspecies was concentrated and injected over an appropriate size exclusioncolumn (Superdex 200) in order to remove aggregates. The SEC poolcontaining all three species (two homodimers and one heterodimer) wassubjected to Immobilized Metal Affinity Chromatography (IMAC) using theNi counter ion under highly discriminating load and elution conditions.The IMAC elution pool contained highly pure heterodimer, with onlyresidual homodimer contamination. IMAC pool buffer was exchanged intoformulation buffer using size exclusion chromatography (Superdex 200),which also removes any residual aggregation products. This IL-21heterodimeric protein was used as a comparator when testing anantibody's neutralizing activity.

Example 3 Preparation of IL-21 Monoclonal Antibodies

Rat monoclonal antibodies are prepared by immunizing 4 femaleSprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.),with the purified recombinant IL-21 protein. The rats are each given aninitial intraperitoneal (IP) injection of 25 μg of the purifiedrecombinant protein in Complete Freund's Adjuvant (Pierce, Rockford,Ill.) followed by booster IP injections of 10 μgof the purifiedrecombinant protein in Incomplete Freund's Adjuvant every two weeks.Seven days after the administration of the second booster injection, theanimals are bled and serum is collected.

The IL-21-specific rat sera samples are characterized by ELISA using 1ug/ml of the purified recombinant IL-21 receptor protein as the specificantibody target. ELISAs comprise preparing IL-21 antigen, coating thewells of a 96-well microtiter plate with the antigen, adding the ratsera of interest to the wells and incubating for a period of time toallow the antibodies in the rat sera to bind to the antigen. A seconddetection antibody (which recognizes the antibodies of interestcontained within the rate sera) conjugated to a detectable compoundconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) is added to thewells. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected as wellas other variations of ELISAs known in the art. For further discussionregarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at11.2.1.

Splenocytes are harvested from a single high-titer rat and fused toSP2/0 (mouse) myeloma cells using PEG 1500 in a single fusion procedure(4:1 fusion ratio, splenocytes to myeloma cells, “Antibodies: ALaboratory Manual”, E. Harlow and D. Lane, Cold Spring Harbor Press).Following 9 days growth post-fusion, specific antibody-producinghybridoma pools are identified by ELISA using 500 ng/ml of therecombinant IL-21 protein as specific antibody target. Positivehybridoma pools are analyzed further for their ability to block thecell-proliferative activity (“neutralization assay”) of purifiedrecombinant IL-21 protein on BaF3 cells expressing the IL-21 receptorsequence.

Hybridoma pools yielding positive results by the “neutralization assay”are cloned at least two times by limiting dilution.

The monoclonal antibodies produced by clones are characterized in anumber of ways including binning (i.e, determining if each antibodycould inhibit the binding of any other binding), relative affinity, andneutralization. Monoclonal antibodies purified from tissue culture mediaare characterized for their ability to block the cell-proliferativeactivity (“neutralization assay”) of purified recombinant IL-21 on Baf3cells expressing the receptor sequences. “Neutralizing” monoclonalantibodies are identified in this manner.

Samples were taken from the hybridoma pools and assayed using both theneutralization assay and a direct titration ELISA. In this assay asample was titrated out using four-fold serial dilutions to see whichclone could maintain the highest OD reading. Using the results from boththe neutralization and titration assays, specific clones from eachinitial master well were chosen to go forward with. Anotherneutralization screen was performed that ran all these samples in thesame assay and at this point the number of cell lines was narrowed downto four top picks. These were subjected to an additional round ofcloning to ensure culture homogeneity and screened using the directELISA. After one more titration assay, two final IL-21 clones werechosen and designated 268.5.1.11.42.1.4.3.9 (rat anti-mouse IL-21, ATCCAccession no. PTA-10394) and 272.21.1.3.4.2 (rat anti-human IL-21, ATCCAccession no. PTA-10395). The monoclonal antibodies produced by thesehybridoma clones can be cultured in a growth medium of 90% Iscove'sModified Dulbecco's medium with 2 mM L-glutamine, 100 mg/mL penicillin,and 100 mg/mL streptomycin sulfate, and 10% Fetal Clone I Serum (HycloneLaboratories). The clones can be propogated by starting cultures at2×10⁵ cells/ml and maintaining between 1×10⁵ and 5×10⁵ cell/ml at 37° C.and 5-6% CO. Cells can be adapted to serum free conditions uponsubsequent transfers. Cells that are frozen are stored in 90% serum, 10%DMSO and stored in vapor phase of liquid nitrogen freezer.

Example 4 Serum Screening of Monoclonal Antibodies

The activity of anti-IL-21 antibodies is measured using a cell-basedpotency bioassay. The bioassay utilizes a BaF3 reporter cell line thatwas engineered to express the IL-21 receptor (IL-21R) through stabletransfection with IL-21R cDNA. The IL-21R/BaF3 transfected cells arehighly dependent upon rIL-21 or IL-3 for growth and, in their absence,are unable to proliferate and undergo apoptosis within 24 hours. In thecell-based bioassay, the IL-21R/BaF3 transfected cells are incubatedwith varying concentrations of serum containing anti-IL-21 antibodiesand subsequent cellular proliferation is measured.

Example 5 Characterization of Antibodies

Epitope Binning

Epitope binning studies are performed on a Biacore1000™ system (Biacore,Uppsalla Sweden). Methods are programmed using Method DefinitionLanguage (MDL) and run using Biacore Control Software, v 1.2. Polyclonalgoat anti-Mouse IgG Fc antibody (Jackson ImmunoResearch Laboratories,West Grove, Pa.) is covalently immobilized to a Biacore CM5 sensor chipand is used to bind (capture) the primary monoclonal antibody of testseries to the chip. Unoccupied Fc binding sites on the chip are thenblocked using a polyclonal IgG Fc fragment (Jackson ImmunoResearchLaboratories, West Grove, Pa.). Subsequently, IL-21 is injected andallowed to specifically bind to the captured primary monoclonalantibody. The Biacore instrument measures the mass of protein bound tothe sensor chip surface, and thus, binding of both the primary antibodyand IL-21 antigen are verified for each cycle. Following the binding ofthe primary antibody and antigen to the chip, a monoclonal antibody ofthe test series is injected as the secondary antibody, and allowed tobind to the pre-bound antigen. If the secondary monoclonal antibody iscapable of binding the IL-21 antigen simultaneously with the primarymonoclonal antibody, an increase in mass on the surface of the chip, orbinding, is detected. If, however, the secondary monoclonal antibody isnot capable of binding the IL-21 antigen simultaneously with the primarymonoclonal antibody, no additional mass, or binding, is detected. Eachmonoclonal antibody tested against itself is used as the negativecontrol to establish the level of the background (no-binding) signal.Data are compiled using BioEvaluation 3.2 RCI software, then loaded intoExcel™ for data processing.

Western Blotting

The ability of the neutralizing monoclonal antibodies from clones todetect denatured and reduced/denatured IL-21 from two sources isassessed using a Western blot format. A rabbit polyclonal antibody knownto detect IL-21 in a Western blot format is used as a positive control.

IL-21 protein is loaded onto 4-12% NuPAGE Bis-Tris gels (Invitrogen,Carlsbad, Calif.) in either non-reducing or reducing sample buffer(Invitrogen) along with molecular weight standards (SeeBlue;Invitrogen), and electrophoresis is performed. Followingelectrophoresis, protein is transferred from the gel, the nitrocelluloseblots are blocked overnight and exposed to each antibody. The blots arethen probed with a secondary antibody conjugated to horseradishperoxidase; sheep anti-mouse IgG-HRP (Amersham: Piscataway, N.J.) forthe monoclonal antibodies and donkey anti-rabbit Ig-HRP (Amersham) forthe polyclonal antibodies. Bound antibody is detected using achemiluminescent reagent (Lumi-Light Plus Reagent: Roche, Mannheim,Germany) and images of the blots were recorded on a Lumi-Imager(Mannheim-Boehringer).

Example 6 DTH Mouse Model

DTH responses are classic immune responses that are initiated by CD4+ Tcells and mediated by T cells, neutrophils and macrophages. A DTHresponse is a good indicator of a CD4+ T cell mediated response. Miceare immunized sub-cutaneously with chicken ovalbumin protein (OVA) ineither of 2 adjuvants, RIBI or CFA. This phase is called thesensitization phase (days 0-6). Ear measurements are taken seven dayslater. Mice are then injected in the ear with control PBS (left ear) orOVA (right ear). This phase is called the challenge phase (days 7-8)Immune responses generated to OVA induce inflammation in the earresulting an increase in ear thickness in 24 hours in the OVA-treated,but not in the PBS-treated ear. This is measured using calipers.

C57BL/6 mice (n=8/group) are immunized in the back with 100 μg chickenovalbumin (OVA) emulsified in RIBI adjuvant (Corixa, Seattle, Wash.) ina total volume of 200 μl. A 0.5 mg/ml of ovalbumin is added to a singlevial of RIBI and vortexed vigorously for 2 minutes to form an emulsionthat is used to inject mice. Seven days after the immunization, mice areinjected with 10 μL1 PBS in the left ear (control) and with 10 μg OVA inPBS in the right ear in a volume of 10 μl. Ear thickness of all mice ismeasured before injecting mice in the ear (0 measurement). Ear thicknessis measured 24 hours after challenge. The difference in ear thicknessbetween the 0 measurement and the 24 hour measurement is calculated andis reflective of the inflammation in the ear. Groups of mice areinjected with PBS or different concentration of anti-IL-21 antibodyintra-peritoneally from either days 0-6 (sensitization phase) or fromdays 7-8 (challenge phase). The injection on day 7 and 8 is given 2hours before measuring ear thickness at the 0 and 24 hour time points.At the end of the 24 hour period, once ear thickness was measured, theears were cut and placed in formalin for histological analysis.

Example 7 Mouse Model for Multiple Sclerosis

To test if anti-IL-21 has any effects on multiple sclerosis, the abilityof anti-IL-21 antibodies to inhibit experimental autoimmuneencephalomyelitis (EAE), a mouse model for MS is tested. The wellcharacterized myelin oligodendrocyte glycoprotein (MOG) 35-55 peptideimmunization model in C57BL/6 mice is used. The experiment is run todetermine that anti-IL-21 antibody could delay and/or inhibit diseasescores in EAE either by inhibiting DC mediated antigen presentation orby enhancing CD8 T cell responses. Absence of efficient CD8 T cellresponses in this model exacerbates EAE (Malipiero et. al., Eur. J.Immunol, 27:3151-3160, 1997). Delayed onset of disease in the EAE modelin a dose dependent manner suggests that use of anti-IL-21 antibody maybe beneficial in MS.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, C57BL/6 mice are immunized with 100 μg MOG pepetide(MOG35-55) or 100 μg recombinant MOG protein emulsified in RIBIadjuvant. Two milliliters of a 0.5 mg/ml preparation of the MOG35-55 inPBS is added to a vial of RIBI and vortexed vigorously to emulsify thesolution or a 1:1 ratio of recombinant MOG in DFA is prepared. The backsof mice are shaved and 100 μg MOG/RIBI is injected s.c in the backs ofmice. Weights of mice are taken 2 days before and every day after theimmunization. Mice are then injected on day 2 i.v. with 200 μL1pertussis toxin (PT), a final concentration of 200 ng/mouse. Mice aremonitored daily for clinical scores. Groups of mice are injected i.p.with 200 μL1 PBS, 100 μg BSA, 10 μg-200 μg anti-IL-21 antibody in a 200μl volume from days 0-20, or 3× a week for 3 weeks. The weights of mice,clinical scores and incidence are evaluated and plotted for analysis.

Example 8 CD4+CD45RBhi (CD25−) Colitis and Psoriasis Mouse Model

Transfer of CD4+ CD45RBhi or CD4+CD25− T cells into syngenic SCID miceresults in colitis in the mice. Co-transfer of regulatory T cells(CD4+CD25+ or CD4+CD45RBlo) inhibits this colitis. After transfer ofCD4+CD25− T cells into mice, if mice are additionally injected withstaphylococcal enterotoxin B (SEB), mice not only develop colitis, butalso psoriasis. Anti-IL-21 antibody is administered from days 0-21 aftercell transfer and symptoms for colitis and psoriasis are monitored.Inhibiton of psoriatic score or colitis (histology) indicates thatanti-IL-21 antibody can inhibit these autoimmune diseases.

Spleens and inguinal lymph nodes are isolated from B10.D2 mice. Singlecell suspensions are formed and counted. Using the Miltenyi Bead system,CD25+ cells are sorted out by positive selection. Cells are stained withCD25−PE (BD Pharmingen) at 1:100 dilution and incubated for 15 minutes.Excess antibody is washed out and the cells are incubated with 10 ulanti-PE beads/106 cells for 20 minutes. The cells are washed with PBSand passed over an LS column (Miltenyi Biotech). Cells that pass throughthe column (CD25) are retained for further analysis. A CD4 enrichmentcocktail (Stem Cell technologies) is added (1:100) to these CD25− cellsand incubated for 15 minutes. Cells are washed with PBS. A 1:10 dilutionof anti-biotin tetramer is added to the cells for 15 minutes followed bya magnetic colloid (60 ul/106 cells) for 15 minutes (all from Stem CellTechnologies). Cells are passed through a negative selection column(0.5″, Stem cell Technologies). Cells that pass through are theCD4+CD25− cells. Purity is analyzed using flow cytometry. 0.4×10⁶ cellsare injected i.v into naïve CB-17 SCID mice in a total volume of 200 μl.Mice are injected i.p with 10 μg SEB the following day (dl). Symptomsfor psoriasis and colitis are followed from 2-5 weeks. Groups of miceare injected i.p. with PBS, 100 μg BSA or 10-200 μg IL-21 from days1-20, or 3× a week for 3 weeks.

Inhibiton of psoriatic and colitis symptoms in anti-IL-21 antibodytreated mice indicates that anti-IL-21 antibodies can inhbit autoimmunesymptoms in this model for psoriasis and colitis.

Example 9 Contact Hypersensitivity Mouse Model

Contact hypersensitivity can be induced in mice using a variety ofcontact allergens including dinitrofluorobenzene (DNFB) and oxazolone.Mice are sensitized topically with the allergen in a vehicle of acetoneand olive oil and then challenged in the ear with the allergen in oliveoil alone. Change in ear thickness is a measure of the immune responseagainst the allergen. Anti-IL-21 antobodies are administered either atthe sensitization phase (d0-5) or during the challenge phase (d5-6).Inhibition of ear thickness by IL-21 indicates a role for IL-21 ininhibiting contact hypersensitivity.

C57B1/6 mice are painted in the back with 0.5% DNFB in acetone:olive oil(4:1) or acetone:olive oil alone on d0. On d5, ear thickness of mice ismeasured using calipers and mice are challenged in the ears with oliveoil alone (control) or 0.25% DNFB in olive oil by dropping a 25 μlsolution onto the ear. Change in ear thickness is measured on d6 and theinflammation calculated as a difference in ear thickness between d5 andd6. Groups of mice are injected i.p. with PBS or 10-100 μg anti-IL-21antibodies on either days 0-5 or days 5-6.

Inhibition of ear thickness by anti-IL-21 antibodies demonstrate thatanti-IL-21 antibodies can be useful in inhibiting contacthypersensitivity.

Splenocytes are harvested and pooled from two high-titer Balb/c mice andfused to P3-X63-Ag8.653 mouse myeloma cells using PEG 1450 in a singlefusion procedure (2:1 fusion ratio, splenocytes to myeloma cells,“Antibodies: A Laboratory Manual”, E. Harlow and D. Lane, Cold SpringHarbor Press). Following 9 days growth post-fusion, specificantibody-producing hybridoma pools are identified by Direct and CaptureELISA using recombinant IL-21 protein, untagged and human IgG Fc tagged,as specific antibody target. Positive hybridoma pools are analyzedfurther for their ability to block the cell-proliferative activity(“neutralization assay”) of purified recombinant IL-21 protein on BaF3cells expressing the IL-21 receptor sequence. Monoclonal antibodiespurified from tissue culture media are characterized for their abilityto block the cell-proliferative activity (“neutralization assay”) ofpurified recombinant IL-21 on Baf3 cells expressing the receptorsequences. “Neutralizing” monoclonal antibodies are identified in thismanner.

Hybridoma pools yielding positive results by the “neutralization assay”and ELISA formats are cloned at least two times by limiting dilution. Inthese assays, samples are titrated using four-fold serial dilutions tosee which clone will maintain the highest OD reading Using the resultsfrom both the neutralization and titration assays, two specific clonesfrom each initial master well are selected for further analysis. Theseare subjected to an additional round of cloning to ensure culturehomogeneity and screened using the Direct ELISA. After one additionaltitration assay, two final IL-21 clones are selected. Hybridoma clonesare cultured in a growth medium of 90% Iscove's Modified Dulbecco'smedium with 2 mM L-glutamine, 100 mg/mL penicillin, and 100 mg/mLstreptomycin sulfate, and 10% Fetal Clone I Serum (HycloneLaboratories). The clones are propagated by seeding cultures at 2×10⁵cells/ml and maintaining between 1×10⁵ and 5×10⁵ cell/ml at 37° C. and5-6% CO. Cells are adapted to serum free conditions upon subsequenttransfers. Cells are frozen in 90% serum, 10% DMSO and stored in vaporphase of a liquid nitrogen freezer.

The purified monoclonal antibodies produced by the hybridoma clones arecharacterized in a number of ways including binning (i.e, determining ifeach antibody could inhibit the binding of any other binding), epitopemapping using peptides, relative affinity, and neutralization.

Example 11 Selection of Peptide Sequences for Use in the Evaluation ofMonoclonal Antibodies Directed Against Human IL-21

An assessment of the binding of murine anti-human IL21 antibodies to thevarious domains of IL-21 was conducted in part through the ability ofthe monoclonal antibodies to bind synthetic peptides derived from thenative human IL-21 sequence. Peptides of 18-29 amino acids were selectedto provide significant coverage of the cytokine polypeptide whilefocusing on domains predicted from cytokine mutein studies and thestructure of IL-21 related cytokines to be important in receptor bindingor activation. Peptides in this size range are also efficient tomanufacture and are of a size that may provide limited secondarystructure for antibody recognition. Peptides 1, 3, and 4 weresynthesized with an amidated carboxyl terminus to better mimic theelectrostatic charge found in the native peptide bonds.

Peptide #1

The N-terminus of human IL-21 following the mammalian processing of thesignal peptide sequence, along with the adjacent amino acid sequence ofIL-21 (SEQ ID NO: 6) was chosen for one peptide. Mammalian expression ofhuman IL-21 and N-terminal sequencing of the cytokine had previouslydemonstrated that following cleavage of the signal peptide, theresulting N-terminal amino acid was the pyroglutamate derivative ofglutamine-30. This derivative was chosen for the N-terminal amino acidfor the peptide and along with the subsequent 20 amino acids found atthe amino terminus of human IL-21. To permit efficient and specificcoupling of the peptide to carrier proteins or solid phase matrixnecessary for the analysis with this peptide, an additional cysteineresidue was added to the carboxyl terminus of the peptide. The completepeptide sequence is pyroGQDRHMIRMRQLIDIVDQLKCamide (SEQ ID NO: 1).

Peptide #2

The second peptide was chosen due to its hydrophilic character, thepresence of proline residues (predicted non-helical segment), and itslocation in the IL-21 sequence between the predicted A- and B-helicalregions. The carboxy terminal end of the peptide was selected due to thepresence of a cysteine residue in the human IL-21 polypeptide sequence.The peptide sequence is NDLVPEFLPAPEDVETNC (SEQ ID NO: 2).

Peptide #3

This peptide sequence was selected for its predicted location comprisinga significant portion of the hydrophilic C-helix of the IL-21 structure.This very hydrophilic region is predicted to be important inligand-receptor interaction, and the peptide span was also selected toallow for the inclusion of a native cysteine residue (Cys-122) to enableefficient conjugations when appropriate as noted above. The peptidesequence is NVSIKKLKRKPPSTNAGRRQKHRLTCamide (SEQ ID NO: 3).

Peptide #4

This peptide was selected due to its location near the carboxyl terminusof the cytokine and because studies utilizing IL-21 muteins havedemonstrated this peptide region to be important for ligand-receptoractivation but not ligand binding. Mutation of Gln-145 and/or Ile-148contained within the sequence of this peptide, have been shown to affecthuman IL-21 receptor activation. The 29 amino acid peptide was initiatedat the native Cys-125 to enable its use in chemical coupling of thepeptide as noted above, and to end at Ser-153. This serine is the finalamino acid in murine IL-21 so the peptide sequence amino terminal ofthis residue is predicted to contain the elements of the human IL-21sequence that are important for ligand activity. The peptide sequence isCDSYEKKPPKEFLERFKSLLQKMIHQHLSamide (SEQ ID NO: 4).

Example 12 Phosphorylated-STAT3 Assay for Detection of IL-21Neutralization

Previously derived Baf3/human IL21 receptor (hIL-21R) transfectants wereused (see, U.S. Pat. Nos. 6,307,024 and 6,686,178, incorporated hereinby reference). The cells were washed three times in Baf3 bioassay mediawhich consists of: RPMI, 1× Glutamax, 10% Fetal Bovine Serum, 50 uMBeta-mercaptoethanol, 200 ug/mL Zeocin, 1 mg/mL G418 (all fromInvitrogen Corporation, Carlsbad, Calif.). After third wash, cells werecounted using standard methods (hemacytometer) and resuspended to 6×10⁵cells per mL in bioassay media. Cells were then plated in a 96-wellround bottom tissue culture plate at 30,000 cells per well. The platewas then transferred to a 37° C. tissue culture incubator while theother assay plates were set up.

The samples plate was then set up with 30 uL of 2.0 ng/mL human IL-21plus 30 uL of one of the following: diluted mouse serum (1:10, 1:50 or1:100 final concentrations), media, anti-IL-21 neutralizing antibody(various lots and concentrations), soluble hIL-21R (example 2) orirrelevant controls. The plate was then transferred to a 37° C.incubator. After 30-40 minutes, both the cell plate and the sampleplates were removed from the incubator and 50 uL of each well in thesample plate was transferred to the cell plate and mixed. The plateswere then placed back in the 37° C. incubator for exactly 8 minutes. Atthis point, the reaction was stopped by placing the plate on ice andadding 150 uL of ice cold BioPlex Cell Wash Buffer (BioRad Laboratories,Hercules, Calif.). The plate was centrifuged for 5 minutes at 1500 RPMand 4° C. Following centrifugation, the supernatant was discarded intothe sink and cells were lysed in 60 uL BioPlex Cell Lysis Buffercontaining Factor 1, Factor 2 and PMSF (all from BioRad). Lysed cellswere pipetted to break up clumps and then shaken at 600 RPM on at 4° C.for 20 minutes. The plate was then centrifuged again for 20 minutes at3000 RPM at 4° C. After centrifugation, 55 uL of lysate was removed andmixed with 55 uL of Phosphoprotein Testing Assay Buffer (BioRad).

At this point a filter plate was pre-wetted with 50 uL PhosphoproteinWash Buffer (PWB), aspirated and 50 uL of PhosphoSTAT3 Coupled Beads(BioRad) plated. These beads were then aspirated and the plate waswashed three times with 75 uL of PWB. Following final aspiration, 50 uLof diluted lysate was transferred to the plate which was then coveredand shaken overnight at room temperature. The following morning, theplate was washed three times with PWB, and biotinylated-PhosphoSTAT3Detection Antibodies (BioRad) were then added for 20 minutes at roomtemperature. The plate was washed three more times in PWB and thenStreptavidin-PE was added for 10 minutes. Finally, the plate was washedthree times with Phosphoprotein Resuspension Buffer (PRB) and the beadswere resuspended in 125 uL of PRB.

Total phosphorylated-STAT3 was measured in each well by following thestandard Luminex 100 data collection protocol as recommended by themanufacturer (Luminex Inc., Austin, Tex.). Data were then analyzed andexpressed as fold-induction of phosphorylated-STAT3 as compared to mediaalone.

Serum from mice immunized with various IL-21 peptides (see table 1) wastested for neutralization of IL21-induced STAT3 phosphorylation. IL-21was premixed with a dilution of serum for 30 minutes at 37° C. Thissolution was then added to Baf3/hIL21R transfectants for 8 minutes. Thereaction was then stopped, cells lysed and phosphorylated-STAT3measured. The fold increase in phosphoylated-STAT3 is calculated usingthe “media alone” control as a baseline. Lower numbers correlate tostronger IL-21 neutralization. The data is summarized in Table 2 below.

Briefly: one of three mice (#1976) immunized with peptide #1 and two ofthree mice (#1979, 1980) immunized with peptide #2, generated IL-21neutralizing antibodies. No mice immunized with peptides #3 and #4generated neutralizing antibodies. The neutralization seen in the 1:10dilution column may be due to a serum effect, not specific anti-IL-21activity (refer to “irrelevant serum” data).

TABLE 1 IL21 peptides and corresponding serum sample numbers Sample #Peptide # 1976-1978 Peptide #1 (see example 11) 1979-1981 Peptide #2(see example 11) 1982-1984 Peptide #4 (see example 11) 1985-1987 Peptide#3 (see example 11) 1988-1990 Peptide #3 (see example 11)

TABLE 2 Average fold increase in phosphorylated- STAT3 levels ascompared to media Serum Dilution 1:10 1:50 1:100 Sample# 1976 1.89 5.075.15 1977 4.98 10.14 8.63 1978 6.72 8.33 12.99 1979 1.04 1.81 2.17 19800.65 1.4 1.81 1981 6.91 9.8 17.06 1982 4.04 10.8 12.73 1983 9.68 12.0512.42 1984 5.84 10.84 11.78 1985 6.94 12.64 11.5 1986 9.01 13.41 16.611987 8.48 14.11 17.36 1988 4.56 8.66 9.38 1989 4.88 11.63 12.66 19909.56 10.29 14.58 Controls Media Alone 1 IL-21 12.4 NMS 11.28 11.72 10.31Irrelevant Serum 3.52 11.09 9.49

Example 13 Assay Description of Direct EIA

The ability of human IL-21 peptides (example 11) to bind anti-humanIL-21 antibodies was assessed using a “direct” style ELISA assay. Inthis assay, wells of 96 well polystyrene ELISA plates were first coatedwith 100 μL/well of human IL-21 protein at a concentration of 1 μg/mL inCoating Buffer (0.1M Na₂CO₃, pH 9.6). Plates were incubated overnight at4° C. after which unbound peptides were aspirated and the plates washedtwice with 300 μL/well of Wash Buffer (PBS-Tween defined as 0.137M NaCl,0.0022M KCl, 0.0067M Na₂HPO₄, 0.0020M KH₂PO₄, 0.05% v/w polysorbate 20,pH 7.2). Wells were blocked with 200 μL/well of Blocking Buffer(PBS-Tween plus 1% w/v bovine serum albumin (BSA)) for 1 hour, afterwhich the plates were washed twice with Wash Buffer. Antibody dilutionswere prepared in 5% FBS/IMDM medium and adjusted to 1 ug/ml. Duplicatesamples of each antibody dilution were then transferred to the assayplates, 100 μL/well, in order to bind anti-human IL-21 peptides.Following 1 hour incubation at RT, the wells were aspirated and theplates washed twice as described above. Horseradish peroxidase labeledGoat anti Mouse IgG, Fc specific (Jackson ImmunoResearch Laboratories,West Grove, Pa.) at a dilution of 1:5000 with 5%/IMDM medium was thenadded to each well, 100 μL/well, and the plates incubated at RT for 1hour. After removal of unbound HRP conjugated antibody, the plates werewashed five times, 100 μL/well of tetra methyl benzidine (TMB) (BioFXLaboratories, Owings Mills, Md.) added to each well and the platesincubated for 3 minutes at RT. Color development was stopped by theaddition of 100 μL/well of 450 nm TMB Stop Reagent (BioFX Laboratories,Owings Mills, Md.) and the absorbance values of the wells read on aMolecular Devices Spectra MAX 340 instrument at 450 nm.

Example 14 Characterization of Neutralizing Antibodies

Characterization of samples derived from hybridoma culture supernatantincluded binding assays using a human IL-21 Fc fusion protein, mouseIL-21 Fc fusion protein, a human IL-21 protein mutated at Gln 145 andIle148 (SEQ ID NO: 6) and peptides described above. IL-21 Fc fusions aredisclosed in U.S. Pat. Nos. 6,307,024 and 6,686,178, and methods forgenerating Fc fusions are disclosed in U.S. Pat. Nos. 5,155,027 and5,567,584, all incorporated herein by reference. The IL-21 mutantprotein is described in U.S. Pat. No. 6,929,932 and U.S. PatentApplication No. 2005-0244930, all incorporated herein by reference.

TABLE 3 Binds Binds hIL-21- Binds mIL-21- Binds Neutral- Fc hIL-21 mFcpeptide Designation Isotype ization protein mutein protein number MouseAb IgG1 Yes Yes Yes Yes No. 3 338.17 Mouse Ab IgG1 Yes Yes Yes Yes No. 3338.24 Mouse Ab IgG1 Yes Yes No Yes None 338.25 Mouse Ab IgG1 Yes YesYes Yes No. 1 338.29 Rat Ab IgG2a Yes No Yes Yes No. 1 272.21.1.3.4.2

Mouse Ab 338.17 This sample bound the human IL-21 protein structure inan area predicted to be in the region of the C-Helix based on itsability to bind Peptide #3.

Mouse Ab 338.24 This sample bound the human IL-21 protein structure inan area predicted to be in the region of the C-Helix-C based on itsability to bind Peptide #3.

Mouse Ab 338.25 This sample bound the D-helix side of the molecule nearQ145 and/or 1148 (SEQ ID NO: 6) based on its ability to bind the nativehIL-21 but not the hIL-21 mutein. The binding is likely to be to adiscontinuous epitope based on its inability to bind peptide #4, whichcontains the native peptide sequence mutated in the IL-21 mutein, or theepitope may require the presence of Ser154-Ser162 of the human IL-21sequence (SEQ ID NO: 6).

Mouse Ab 338.29 This sample bound the human IL-21 protein structure inan area predicted to be in the region of the A-Helix based on itsability to bind Peptide #1. Based on its ability to react moderatelywith a C-terminally conjugated Peptide #1 but react very strongly to thenon-conjugated peptide, one would predict the epitope for this antibodyto include the middle or C-terminal domain of Helix A as represented inPeptide #1.

Rat Ab 272.21.1.3.4.2 Based on its ability to react weakly with both theC-terminally conjugated and non-conjugated Peptide #1, its ability toneutralize hIL-21 but its inability to capture hIL-21-Fc from solution.,this antibody binds to a largely discontinuous epitope on the humanIL-21 protein structure comprising an area predicted to be in the regionof the N-terminus of IL-21 and the A-Helix and require space near theadjacent C-terminus of hIL-21 where it is linked to the Fc fusionprotein.

1. An isolated antibody that is capable of binding to the same epitopeas a monoclonal antibody produced by the hybridoma designated ATCCAccession No. PTA-10395 for binding a human interleukin (IL)-21 antigen.2. The isolated antibody of claim 1, wherein the antibody neutralizesthe protein activity of human IL-21.
 3. The antibody of claim 1, whereinsaid antibody is humanized.
 4. An isolated antibody that is capable ofbinding to the same epitope as a monoclonal antibody produced by thehybridoma designated ATCC Accession No. PTA-10394 for binding a murineinterleukin (IL)-21 antigen.
 5. The isolated antibody of claim 4,wherein the antibody neutralizes the protein activity of human IL-21. 6.The antibody of claim 4, wherein said antibody is humanized.